Intestinal epithelial cell (IEC) gene expression must be tightly regulated to avoid over-reaction to ubiquitous bacterial products, yet be able to adequately respond to environmental pathogens. Developing evidence suggests that chronic intestinal inflammation is the result of a genetically determined, overly aggressive mucosal immune response to normal resident luminal bacterial components. Although the repertoire of pro-inflammatory molecules synthesized by IEC has been identified, little is known about how these cells regulate expression and suppression of these genes. Moreover, the intracellular signaling pathways associated with IEC gene expression are still poorly understood. Most of the molecules involved in the immune and inflammatory response are in part controlled at the transcriptional level by a coordinated group of transcription factors. We have shown that the IkappaB/NFkappaB family of transcription factors regulates expression of a number of pro-inflammatory cytokines and adhesion molecules in IEC and that native IEC and most transformed colonic cell line show a unique IkappaBalpha phosphorylation and degradation process that distinguishes these cells from other cell lineages (1). Our hypothesis is that normal IEC have a unique IkappaB/NFkappaB regulatory system buffering their cellular responses to the aggressive luminal environment. This decreased responsiveness to ubiquitous microbial stimuli and pro-inflammatory cytokines helps maintain mucosal homeostasis and is important in the protective responses which restore normal balance following intestinal injury and inflammation. This hypothesis will be addressed by the following SPECIFIC AIMS: 1) Identify the unique mechanisms regulating activation of the IkappaB/NFkappaB transcriptional system in transformed and native IEC following stimulation with cytokines, bacteria and bacterial products, 2) Determine the molecular mechanisms of corticosteroid blockade of IkappaB/NFkappaB activation in IEC, and 3) Identify the functional consequence of selectively modulating components of the IkappaBalpha/NFkappaB system. A better understanding of activation and regulation of this central signaling pathway in the response of intestinal epithelial cells to environmental stimuli will provide new insights into the molecular mechanisms of mucosal homeostasis and inflammation and suggest new targets for therapeutic blockade of NFkappaB activation.